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What to do when your Plates Fail
If your E-gel comes out completely blank (not even one band) you may have made a mistake somewhere along the way (ie. Subsampling, Lysis, extraction, adding DNA to PCR). #The primer that was used may not be ideal for the organism that is being sequenced. A different primer could be used. Mammal cocktail often works for problematic fish samples. For insects, Folmer or LepF/R can be used. There are also specific primers for crustaceans and others for skates. #You might try to optimize the reaction by adding different amounts of MgCl2 to the PCR reaction. Try adding various amounts to the samples, (try somewhere between 0.2ul to 0.6) and then running the PCR. #If you are working with old specimens, the DNA is most certainly degraded. If you only come out with a few bands after using regular PCR primers, try it again with mini-primers. If that still does not work, do not despair. #Using the nano-drop machine will tell you if you have DNA in your sample. If the concentration is too high or too low the PCR may not work well. Instructions can be found at Using the Nano Drop. #If you end up with very low amounts of DNA, then you can reamplify your PCR product in another reaction. Even though the reaction failed to show bands on the E-gel, there is still PCR product in it. Use the 1ul of PCR product from a plate with full-length primers in a plate with mini-primers or even will full-length primers. #If you end up with a high concentration of DNA, then the PCR may have failed because the DNA was too concentrated. Run a PCR reaction using 1ul of DNA. #If you end up with bright smears rather than nice bands, your DNA may be incredibly concentrated. Try diluting 2ul DNA in 10ul ddH2O and use 2ul of that in another PCR. The bright smear is the genomic DNA. #You can have your plates positive hit-picked in BIO. This means that a robot will take two (or more) plates that each had less than 85 bands and pipette the PCR product out from each plate and put them together to form a plate with 96 samples that all had bands on the E-gel. If you only have one plate that you are working on, tell the lab manager at BIO who can hit pick the plate with another partial plate that is in BIO. #Negative hit picking is when you remove the samples that failed to another plate. For example, if you had ten plates and each plate had ten or fifteen samples that failed, you would use negative hit picking to put the DNA from the failed samples together into one plate, so that you can try PCR reactions only on the samples that failed, and not waste reagents on samples that worked initially. #The amount of primer that you are using may need to be adjusted. BIO uses 0.125ul of forward or reverse primer per sample. We have found that this is at times too much and forms primer dimers which make a second band on the gel that is fainter but then it will affect sequence quality, so using 0.075ul can help cut that down. But using not enough primer may cause the reaction to not produce a band, so for this reason I have found that 0.100ul of primer to work best. #If you are testing with 16s to see if there is any DNA at all, I have found that you need to PCR the PCR each time to make bands show. Return to Barcoding in the Hanner Lab Wiki. Return to PCR Procedure. Updated June 25 2009